ZBiotech’s Glycosaminoglycan Array and Heparan Sulfate Glycan Array facilitated the groundbreaking discovery of novel glycan ligands for human VSIG4.

Leveraging the power of our advanced glycan arrays, esteemed scientists were able to unravel the intricate interactions between heparan sulfates and VSIG4, shedding light on the preferential binding of highly sulfated moieties and longer GAG chains. These findings suggest a crucial contribution of these factors to the specificity of this interaction, opening new avenues for exploration.

The landscape of protein-glycan interactions remains largely uncharted, harboring immense potential for future breakthroughs. ZBiotech stands at the forefront of this scientific frontier, offering an exceptional glycan array platform that empowers researchers to illuminate the fascinating realm of glycobiology in human health and disease. Our state-of-the-art arrays present an invaluable toolset, empowering scientists to unravel the complex language of glycans and decipher their intricate roles in various biological phenomena.

 

Exploring the Role of Heparan Sulfates as Novel Glycan Ligands for VSIG4

In a recent article published in Glycobiology, the scientists discovered a novel interaction between V-set and immunoglobulin domain-containing 4 (VSIG4) and heparan sulfates, a type of glycosaminoglycan (GAG).

VSIG4 is known to be expressed on tissue-resident macrophages and has been implicated in various immune functions and disease phenotypes. The study revealed that heparan sulfates, both cell-surface and soluble forms, are novel binding partners of VSIG4. By utilizing enzymatic removal assays, competition assays, genetic studies, glycan array analysis, and direct binding assays, the researchers established a direct interaction between VSIG4 and heparan sulfates. They found that heparan sulfates compete with known VSIG4 binding partners, C3b and iC3b, indicating a potential regulatory role for heparan sulfates in VSIG4 biology. However, soluble heparan sulfates did not affect phagocytosis mediated by VSIG4, suggesting potential differential effects of cell-anchored and soluble heparan sulfates on VSIG4 functions.

The affinity between VSIG4 and heparan sulfates was found to be within the range of other reported heparan sulfate-protein interactions. Interestingly, the affinity was observed to be higher for mouse VSIG4 compared to human VSIG4, possibly due to differences in the electrostatic potentials between the IgV domains of the two species. Furthermore, the long form of human VSIG4 exhibited a higher affinity for heparan sulfates compared to the short form, suggesting the involvement of the IgC2 domain in enhancing the binding affinity. The study also demonstrated that VSIG4 prefers highly sulfated moieties and longer heparan sulfate chains. Mutagenesis studies revealed that positively charged amino acids within the heparan sulfate-interacting epitope were critical for the VSIG4-heparan sulfate binding.

Heparan sulfates are known to have diverse effects on the immune system, both inhibiting and promoting inflammation. The researchers speculated that the VSIG4-heparan sulfate interaction may play an immunomodulatory role and contribute to inflammation regulation. The precise in vivo context and functional consequences of the VSIG4-heparan sulfate interaction requires further investigation.

These findings shed light on a novel interaction between VSIG4 and heparan sulfates and provide insights into the regulation of VSIG4-associated functions. The study also highlights the importance of understanding the impact of heparin and heparan sulfate mimetics on VSIG4-complement interactions, particularly in the context of therapeutic development. Overall, this research contributes to our understanding of the complex role of VSIG4 in immune regulation and uncovers a previously unrecognized interaction between VSIG4 and heparan sulfates, opening avenues for further investigation into their physiological significance and therapeutic implications.

 

How does our glycan array technology facilitate the determination of direct binding between VSIG4 and heparan sulfates?

Utilizing an efficient assay that completes in about four hours, the interactions between VSIG4 and heparan sulfates were elucidated. Both mVSIG4-fc and hVSIG4-fc demonstrated binding to heparan sulfate glycans that carried sulfate modifications. This finding aligns with the FACS and SPR data, thereby validating the reliability of our glycan array technology in precisely determining glycan-protein interactions.

 

Given that our heparan sulfate glycan array encompasses 24 structurally defined heparin and heparan sulfate glycans, varying in length and sulfation, scientists were able to further discern a positive correlation between the number of sulfate groups per disaccharide and the binding signals. This observation potentially suggests that the sulfation pattern and the length of the heparan sulfate chain play a decisive role in their interaction with VSIG4. This hypothesis was further confirmed by a subsequent competitive binding assay.

 

The process of analyzing glycan-protein interactions using a glycan microarray is quick and intuitive, and it does not necessitate any specialized training.

1️ The initial step involves inserting the microarray slide into a microarray hybridization chamber, a device included in the array kit. Subsequently, the array slide is blocked with Glycan Array Blocking Buffer (GABB) at room temperature for 30 minutes on a rotating shaker. Following the blocking phase, the array slide is washed using Glycan Array Assay Buffer (GAAB).

2️ The VISG4 protein sample, prepared in GAAB, is then added to the slide chamber and incubated for 1 hour at room temperature. Post-incubation, the array slide is rinsed, and an anti-human IgG Fc (Cy3) antibody, prepared in GAAB, is added to the slide, followed by a further incubation for 1 hour at room temperature.

3️ Finally, after washing the slide, it is scanned to reveal the results.